COVID-associated pernio: Mechanistic insights to an abortive, seronegative SARS-CoV-2 infection

Introduction: SARS-CoV-2 replicates primarily in the airways but generates a systemic immune response mediated by Type I interferons (IFN-I). Pernio is a rare skin manifestation of disorders characterized by excessive IFN-I signalling. Although pernio increased in incidence during the pandemic, the relationship to SARS-CoV-2 remains controversial. Because of the pivotal nature of interferons in COVID-19 outcomes, pernio offers a window to investigate the biology underlying host resiliency to SARS-CoV-2 infection. Method(s): To further assess COVID-associated pernio, we characterized clinical samples from affected patients across 4 waves of the pandemic and investigated mechanistic feasibility in a rodent model. Patients were followed longitudinally with banking of blood and tissue. Golden hamsters were mock-treated or intra-nasally infected with SARS-CoV-2 and harvested at 3-and 30-days post-infection. Result(s): In affected tissue, immunophenotyping utilizing multiplex immunohistochemistry profiled a robust IFN-1 signature characterized by plasmacytoid dendritic cell activation. Viral RNA was detectable in a subset of cases using in situ hybridization for the SARS-CoV-2 S gene transcript. Profiling of the systemic immune response did not reveal a durable type 1 interferon signature. Consistent with previous literature, antibody and T-cell specific responses to SARS-CoV-2 were not detected. Nasopharyngeal SARS-CoV-2 inoculation in hamsters resulted in rapid dissemination of viral RNA and the generation of an IFN-I response that were both detectable in the paws of infected animals. Conclusion(s): Our data support a durable local IFN signature, with direct evidence of viral SARS-CoV-2 RNA in acral skin and suggest that COVID-associated pernio results from an abortive, seronegative SARS-CoV-2 infection.

Macrophage phagocytosis of SARS-CoV-2-infected cells mediates potent plasmacytoid dendritic cell activation.

Early and strong interferon type I (IFN-I) responses are usually associated with mild COVID-19 disease, whereas persistent or unregulated proinflammatory cytokine responses are associated with severe disease outcomes. Previous work suggested that monocyte-derived macrophages (MDMs) are resistant and unresponsive to SARS-CoV-2 infection. Here, we demonstrate that upon phagocytosis of SARS-CoV-2-infected cells, MDMs are activated and secrete IL-6 and TNF. Importantly, activated MDMs in turn mediate strong activation of plasmacytoid dendritic cells (pDCs), leading to the secretion of high levels of IFN-α and TNF. Furthermore, pDC activation promoted IL-6 production by MDMs. This kind of pDC activation was dependent on direct integrin-mediated cell‒cell contacts and involved stimulation of the TLR7 and STING signaling pathways. Overall, the present study describes a novel and potent pathway of pDC activation that is linked to the macrophage-mediated clearance of infected cells. These findings suggest that a high infection rate by SARS-CoV-2 may lead to exaggerated cytokine responses, which may contribute to tissue damage and severe disease.

ICAM-1 MEDIATES PLASMACYTOID DENDRITIC CELL SENSING OF SARS-CoV-2-INFECTED CELLS

Background: Plasmacytoid dendritic cells (pDCs) are the major producer of type I IFNs (IFN-I), the critically important antiviral cytokines against SARS-CoV- 2. Although pDCs can sense cell-free SARS-CoV-2 virions, it is unknown whether they can detect infected cells to produce IFN-I. Since cell-to-cell transmission accounts for 90% of SARS-CoV-2 infections (Zeng et al., 2022), we examined the relevance of pDC sensing of infected cells in SARS-CoV-2 infection and whether the virus exploits this pathway to evade IFN-I responses. Method(s): LSPQ1, the first SARS-CoV-2 clinical isolate received from the Public Health Laboratory of Quebec, was used as a prototype virus. SARS-CoV-2 variants of concerns (VOCs) were also used. PBMCs or enriched pDCs were cocultured with mock-infected or SARS-CoV-2-infected HeLa-hACE2 or Calu-3. Either PBMCs, enriched pDCs, or HeLa-hACE2 were pretreated with anti-human ICAM-1 antibody or isotype control. The conjugate formation was determined by flow cytometry. Polarized Caco cells were used to validate critical data. Result(s): Upon sensing infected cells, PBMCs release 6-fold more IFN-I than they do when exposed to cell-free virions. Antibody-mediated depletion of pDCs from PBMCs abolishes IFN-I secretion. Direct contact of pDCs with infected cells is required for sensing since the use of a transwell membrane reduces IFN-I release by 85%. Infected cells form conjugates with pDCs more frequently (3.2-fold higher) than uninfected cells. Blocking ICAM-1 on infected cells or pDCs impacts conjugate formation and significantly suppresses IFN-I production by 55-80%, suggesting bidirectional interaction. Moreover, human lung cells infected with VOCs are sensed to a different extent with the alpha variant being the least efficiently sensed by pDCs compared to the delta or omicron strains. Even though SARS-CoV-2 is primarily released from the apical domain of polarized infected Caco cells, sensing of infected cells does occur upon direct contact of pDCs with the basolateral domain, highlighting how pDCs antiviral responses might be triggered in respiratory tissues. Conclusion(s): pDC sensing of infected cells accounts for the vast majority of IFN-I released during SARS-CoV-2 infection. ICAM-1 promotes physical contact between pDCs and infected cells, thus leading to efficient sensing. Differential pDC sensing of SARS-CoV-2 VOC-infected cells suggests that some VOCs might manipulate the interactions of pDCs with infected cells to limit IFN-I responses.

Impact of Azithromycin and 8-Hydroxychloroquine in 2019 Novel Coronavirus (COVID-19) Pandemic: A Systematic Review

As we know novel coronavirus is an emergent nuisance in this stipulated period. Corona virus is a group of enveloped viruses, with non-segmented, single stranded & positive sense RNA genomes. Human Corona virus is mainly subdivided into four categories such as 229E, NL63, OC43, HKU1. Epidemiologically it has a greater prevalence in the modern era. The features encountered in the clinical course of the disease are multifarious spanning from cough, sneezing, fever, breathlessness. It may take 2-14 days for a person to notice symptoms after infection. Azithromycin and 8 Hydroxychloroquine both plays an instrumental role for management of COVID-19. Azithromycin is a macrolide antibiotic and it binds with a 50s ribosome then inhibits bacterial protein synthesis. On the other hand 8-Hydroxychloroquine was approved by United State in the year of 1955 .Basically it is used as a antimalarial drugs . Briefly, in inflammatory conditions it binds with toll like receptor & blocks them. 8- hydroxychloroquine increases lysosomal pH in antigen presenting cells . In inflammatory conditions it blocks toll like receptors on plasmacytoid dendritic cells. In our review we focused on the role of Azithromycin, and 8-hydroxychloroquine in Covid-19 .Copyright © 2021 International Journal of Pharma and Bio Sciences. All rights reserved.

Enhanced TLR7 immunity drives innate protection against SARS-CoV-2 with chilblains as collateral damage

Outbreaks of chilblains, a hallmark sign of type I interferonopathies, have been reported during the COVID-19 pandemic. These cases occurred mostly in patients who were asymptomatic and showed negative results from PCR and serology tests for SARS-CoV-2. We hypothesized that chilblain patients are predisposed to mount a robust innate immunity against the virus, which clinically manifests as chilblains and promotes early viral clearance, thereby preventing pulmonary disease and precluding adaptive responses. By profiling skin lesions in the early stage following chilblain onset, we uncover a transient IRF7-dependent type I interferon (IFN) signature that is driven by the acral infiltration of systemically activated plasmacytoid dendritic cells (pDCs). Patients' peripheral blood mononuclear cells (PBMCs) demonstrate increased production of IFNalpha when exposed to SARS-CoV-2 and influenza A, but not herpes simplex virus 1 (HSV-1), indicating a heightened ability to detect RNA -but not DNA- viruses. Further investigations revealed enhanced responsiveness of pDCs in chilblain patients to the RNA sensor TLR7, but not the DNA sensor TLR9. Collectively, our study establishes a two-step model for the immunopathology of SARS-CoV-2-related chilblains: enhanced TLR7 immunity in pDCs, likely triggered by SARS-CoV-2 exposure at the mucosal site, leads to prompt viral clearance, which explains the lack of infection markers in most cases. Subsequently, systemic spread of activated pDCs and infiltration of the toes in response to mechanical stress or acral coldness, may result in IFN-mediated tissue damage with development of chilblains.Copyright © 2023

Kikuchi-Fujimoto disease type lymph node reaction with increased plasmacytoid dendritic cells may appear as a side effect following COVID-19 vaccination: Report of a case and literature review

Subsequent to mass vaccination programs against COVID-19, diverse side effects have been described, both at the injection site, such as pain, redness and swelling, and systemic effects such as fatigue, headache, muscle or joint pain. On rare occasions, a lymphadenopathic syndrome may develop, raising the clinical suspicion of a lymphoproliferative disorder. We present the case of a 30-year-old woman who developed self-limiting left axillary lymphadenopathy following COVID-19 vaccination. To date, only seven similar cases with a complete clinicopathological description have been published, and fourteen cases have been notified to the European adverse events databases (Eudravigilance) in relationship with vaccination against COVID-19. It is important to be aware of this potential complication when a lymphadenopathic syndrome develops following vaccination, to avoid unnecessary treatment.Copyright © 2023 Sociedad Espanola de Anatomia Patologica

Involvement of the chemerin-ChemR23 system in severe COVID-19 patients

Background: ChemR23 knock-out mice displays aggravated viral pneumonia, with similar features as observed in severe COVID-19 patients. Aims and objectives: We evaluated the involvement of the chemerin-ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic role. Method(s): Blood samples from confirmed COVID-19 patients were collected at day 1, 5 and 14 from admission to Erasme Hospital (Brussels - Belgium). Chemerin concentrations and inflammatory biomarkers were analyzed in the plasma. Blood cells subtypes and their expression of ChemR23 were determined by flow cytometry. The expression of chemerin and ChemR23 was evaluated on lung tissue from autopsied COVID-19 patients by immunohistochemistry (IHC). Result(s): 21 healthy controls (HC) and 88 COVID-19 patients, including 40 in intensive care unit (ICU) were included. The concentration of chemerin in plasma was significantly higher in ICU patients vs HC at any time-point (p<.0001) and also when comparing deceased patients vs survivors (p=.02). In line with that, chemerin levels correlated with inflammatory biomarkers such as C-reactive protein, interleukin-6 and tumour necrosis factor alpha. Plasmacytoid dendritic cells and natural killers (NK) cells were strongly decreased in hospitalized and ICU COVID 19 patients. On NK cells of all COVID 19 patients, the expression of ChemR23 was reduced regardless its severity. Moreover, IHC analysis showed a strong expression of ChemR23 on smooth muscle cells and chemerin on myofibroblasts during the organizing phase of acute respiratory distress syndrome (ARDS). Conclusion(s): Chemerin is an early marker of severity in COVID-19 patients and could be involved in lung fibrosis post-ARDS.

Correlation between Type I Interferon Associated Factors and COVID-19 Severity.

Antiviral type I interferons (IFN) produced in the early phase of viral infections effectively inhibit viral replication, prevent virus-mediated tissue damages and promote innate and adaptive immune responses that are all essential to the successful elimination of viruses. As professional type I IFN producing cells, plasmacytoid dendritic cells (pDC) have the ability to rapidly produce waste amounts of type I IFNs. Therefore, their low frequency, dysfunction or decreased capacity to produce type I IFNs might increase the risk of severe viral infections. In accordance with that, declined pDC numbers and delayed or inadequate type I IFN responses could be observed in patients with severe coronavirus disease (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as compared to individuals with mild or no symptoms. Thus, besides chronic diseases, all those conditions, which negatively affect the antiviral IFN responses lengthen the list of risk factors for severe COVID-19. In the current review, we would like to briefly discuss the role and dysregulation of pDC/type I IFN axis in COVID-19, and introduce those type I IFN-dependent factors, which account for an increased risk of COVID-19 severity and thus are responsible for the different magnitude of individual immune responses to SARS-CoV-2.

IMMUNE PARAMETERS ASSOCIATED with LOWER SARS-CoV-2 VACCINATION RESPONSE in OLD PEOPLE

Background: The number of cases of SARS-CoV-2 infection after BNT162b2 mRNA vaccination is significantly higher in elderly people, which has been associated to lower frequencies of SARS-CoV-2 neutralizing antibodies. Our objective was to investigate the differences in the cellular response in old and young people after the SARS-CoV-2 vaccination. Methods: Young (24-53 years, n=20) and old (70-76 years, n=20) healthy subjects vaccinated with BNT162b2 SARS-CoV-2 mRNA vaccine were studied before vaccination, two weeks after the first dose and two months after the second dose. SARS-CoV-2 (spike) specific T cell response, TLR-4 dependent monocyte response and TLR-3 dependent myeloid dendritic cell (DC) response and DC, monocyte and T-cell immunophenotype, were studied by multiparametric flow cytometry. TLR-9 dependent interferon-α (IFNα) production by PBMCs was measured by ELISA and thymic function assayed by sj/β TREC ratio using droplet digital PCR. Results: The SARS-CoV-2 specific T cell response was lower and less polyfunctional in old people. Most of the differences in CD4+ and CD8+ T cell subsets were found in degranulation (CD107a), cytokine (IFN-γ) and cytotoxic (perforin) profile (eg, Memory CD8+ perforin+;p=0.0016). The lower SARS-CoV-2 specific T cell response was associated with lower thymic function levels (eg, Memory CD4+ perforin+, r=0.631;p=0.0001). The vaccination induced a higher activation and proliferation (eg, CM CD4 HLA-DR+ p=0.002, Ki67+ p=0.019) of T cells in young people than in old ones, in addition to a higher level of homing makers to different tissues and inflammatory sites (eg, CD1c mDC integrin β7+ p=0.001, intermediate monocytes CCR2+ p=0.0003) in DCs and monocytes. Moreover, after the vaccination, old subjects showed a higher production of proinflammatory cytokines by monocytes in response to LPS (eg, IL6+;p=0.015), while young people showed a higher production of IFNα by plasmacytoid DCs after CpG-A stimulation (p=0.0009). Conclusion: The magnitude and polyfunctionality of SARS-CoV-2 specific T cell response is lower in old people, associated to a lower thymic function. In old people, the vaccination induced less immune activation and homing and the myeloid TLR-dependent response is directed towards a proinflammatory response, while in young people prevails IFNα production, related to a more effective antiviral response. These results support the additional boosting strategies in this vulnerable population.

Histopathological analysis of skin reactions after coronavirus disease 2019 vaccination: Increment in number of infiltrated plasmacytoid dendritic cell.

Skin disorders are frequent adverse events after coronavirus disease 2019 (COVID-19) vaccination. However, the pathogenesis of these disorders is not fully understood. Here, we report a case series of cutaneous adverse events following COVID-19 vaccination, and the results of our investigation reveal the underlying mechanism. Case 1: a 47-year-old female developed a wheal, confined to the COVID-19 vaccination site, 2 days after her first injection. She was treated with topical steroids and oral antihistamines. Case 2: a 51-year-old female showed generalized petechial erythema accompanied by fever, genital bleeding, thrombocytopenia, liver dysfunction, and disseminated intravascular coagulation, 2 days after her second injection. She was diagnosed with vaccine-induced macrophage activation syndrome and treated with anti-inflammatory therapy. Immunohistological analysis of the skin eruption, in both these cases, showed infiltration of CD123+ BDCA2+ plasmacytoid dendritic cells (p-DC). Despite the distinctive clinical features in these two cases, this finding suggests that p-DC might be involved in different cutaneous adverse events after COVID-19 vaccination.

Early Immune Profile Assessment By Flow Cytometry Predicts Severe COVID-19 Pneumonia

[Formula presented] Background and Objective The rapid spread of the COVID -19 pandemic and the high variability of the course of the disease make it essential to search for early predictors of outcome. The objective of our study is to predict severe SARS COV2 pneumonia using early cytometric profiles Material and Methods Prospective and observational study of adults with confirmed COVID-19 infection admitted on Emergency Department (ED). We collected epidemiological, clinical and laboratory data of every patient until they were discharged or died. Multiparametric flow cytometry (FC) analysis of T-lymphocytes (CD4, CD8, CD4 activated, CD8 activated, naïve (Tn), central-memory (Tcm), effector-memory (Tem), effector (Te) and Th17 subsets), B-lymphocytes (naïve, memory, transitional subsets, and assessment of clonality), NK cells, plasmablasts, p-DCs (plasmacytoid dendritic cells), m-DCs (myeloid dendritic cells), basophils, and monocytes (MO1, MO2, MO3, slan+ MO3) was performed on whole peripheral blood collected on EDTA, before immunosuppressive therapy was started. We designed a 7-tube 8-color experimental panel. Cell surface staining of 2 × 106 cells was performed and at least 500 000 total events were acquired for the assessment of plasmablasts, p-DCs, m-DCs, basophils, and the monocyte subsets;for the study of B, T and NK-lymphocyte populations we acquired at least 100 000 total events (FACS Canto II;BD Biosciences). Severity was assessed on the basis of World Health Organization´s (WHO) international 10 level ordinal scale (WHOs) and also according to 4 respiratory status, based on SpO2(peripheral blood oxygen saturation)/FIO2 (fraction of inspired oxygen) ratio (SpFi). SpFi group 1 >452, SpFi2 2: 315-452;SpFi 3 236-315, SpFi 4: <236 (respiratory distress) Results 53 patients were included: epidemiological and clinical data available on table 1. 23 patients (43.39%) arrived to SpFi4 status. WHOs >6 (WHO 6:oxygen by non invasive ventilation or high flow) was achieved by 20 patients (37.7%) Good prognosis (meaning SpFi1 as the worse respiratory status in the follow -up) was associated to cytometric profiles: there was a significant increase in CD3, p-DCs, m-DCs, basophils, monocytes, Tcm, % of lymphocytes and CD3/CD19 ratio whereas there was a significant reduction in CD19, % of neutrophils and % Neutrophils/% lymphocyte ratio. In the SpFi4 group, there was a significant reduction of CD3, p-DCs, m-DCs, plasmablasts and CD3/NK ratio. In patients starting in SpFi group 1-2 in the ED but progressing to SpFi 3-4 during the follow up (27 patients), there was a statistically significant relation with Tn, Te and Tn/Te ratio (Tn/Te ratio <0.717: OR 13.5 (p 0.002, [95% CI 2.552-71.403). Initial SpFi1 patients that evolved to SpFi 3-4 during follow up (10 patients), presented a Tn/Te ratio < 0.717 with an OR 11.556 (p =0.005, [95% CI 2.059-64.853]). Plasmablasts < 0.075 and CD3/NK <5.71, were identified as independent risk factors for SpFI4 during follow up. After multivariable analysis, both variables kept their significance: CD3/NK (OR 11,247, p=0.005) and plasmablasts (OR 12,524, p=0.004). About prediction of WHOs >6, multivariable analysis showed CD3/NK <5.71 (OR 22,240 [95%CI 2,340-211,342] p= 0.007) and plasmablasts<0.075 (OR 28.635 [95% CI 3,187-257,301] p=0.003). A score (0,1,2) comprising both risk factors, was significantly predictive of SpFI4, regardless of the initial respiratory status, age or days from symptoms onset. In our cohort, only 1/15 (6.7%) patients with 0 points (neither plasmablasts nor CD3/NK score), arrived to SpFi4. However, 10/11 (90.9%) patients with 2 points, reached to SpFi4 respiratory status (C-index = 0.837) Same score was applied to predict WHOs > 6: 90.9% with 2 points progressed to WHO>6 and 0/15 patients with 0 points reached the same goal (C-index = 0.872) An incidental finding of 4 indolent B-lymphoproliferative disorders (2CLL-like MBLs and 2non -CLL-like MBL), was found, and they were associated with older age and progression to death. Conclusions Flow cytometry on whole pe ipheral blood samples of SARS-COV2 pneumonia patients, collected before corticosteroid or immunosuppressive therapy, could identify cytometric patterns associated to prognosis. Plasmablasts, mDCs and pDCs levels as well as CD3/NK ratio, are associated to a worse respiratory status, while Tn/Te ratio could detect non-severe patients who will require high-flow oxygen devices during follow up. [Formula presented] Disclosures: No relevant conflicts of interest to declare.

Kikuchi-Fujimoto Disease: A Case Report

[Formula presented] Kikuchi-Fujimoto: A Case Report Hickman, JD. MD LT MC USN and An, Joseph, DO. LCDR MC USN Naval Medical Center Portsmouth 620 Johns Paul John Cir, Portsmouth VA 757-953-2223 The views expressed in this are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the United States Government. We are military service members and employees of the U.S. Government. This work was prepared as part of my official duties. Title 17 U.S.C. 105 provides that “Copyright protection under this title is not available for any work of the United States Government.” Title 17 U.S.C. 101 defines a United States Government work as a work prepared by a military service member or employee of the United States Government as part of that person's official duties. Kikuchi-Fujimoto disease is a rare benign disorder often presenting with tender cervical lymphadenopathy, fever, and malaise. While first described in Japan, its distribution is worldwide and predominantly seen in young adults. Diagnosis is based on characteristic histopathologic findings of patchy necrosis occupied by karyorrhectic debris and abundant histiocytes on node biopsy. The origin is unclear but associated with a preceding viral illness as well as cutaneous lupus erythematosus. Treatment is generally supportive and focused on managing tender lymph nodes. We present a case of a 28-year-old female presenting with a 2 month history of night sweats, fever, and weight loss in the setting of painful neck swelling. CT and PET/CT imaging demonstrated numerous hypermetabolic and enlarged nodes in the bilateral cervical and axillary regions. Lab studies were notable for leukopenia, anemia, and elevated inflammatory markers. A COVID-19 screening was negative. Excisional biopsy of a cervical node revealed extensive cortical necrosis and apoptotic debris with scattered histiocytes and plasmacytoid dendritic cells in absence of neutrophils or a monoclonal B cell or T cell population. Treatment was initiated with NSAIDs and close monitoring. The patient exhibited a complete response after two months. Our case is an important reminder that lymphadenopathy, fever, and night sweats in a young adult are not pathognomonic for lymphoma. Nonetheless, a high suspicion for lymphoma should be maintained and followed with an expedited workup. Kikuchi-Fujimoto can certainly mimic Hodgkin lymphoma or other serious conditions like lupus erythematosus and tuberculosis. The diagnosis is largely one of exclusion following a careful examination of a lymph node histopathology and must be considered in young previously healthy adults to avoid misdiagnosis and unnecessary escalation of treatment. Disclosures: No relevant conflicts of interest to declare.